Nucleoside analogue Reverse Transcriptase Inhibitors (NRTIs) are an essential component of HAART (Highly Active AntiRetroviral Therapy), substantially reducing the morbidity and mortality of HIV-1 infection. However, the use of many of these drugs, including ddC, ddl and d4T has been associated with a painful sensory polyneuropathy (Antiretroviral Toxic Neuropathy, ATN) that often necessitates drug discontinuation by the patient. There has been no reliable in vitro or in vivo animal models of NRTI neurotoxicity. We propose to develop a robust and reliable in vitro model of NRTI neurotoxicity to study the cellular mechanism of toxicity and initiate screening of preventative or therapeutic drugs. Dorsal root ganglion (DRG) sensory neurons are the main targets for NRTI toxicity in humans. Therefore, primary DRG sensory neurons from rats will be used to develop this model. We will examine the effects of various NRTIs that are known to cause ATN in humans. Established morphological criteria will be used as primary outcome measures of neurotoxicity. Additionally we will develop biochemical and molecular assays of neurotoxicity using the expression of axonal outgrowth associated genes. These will be validated against the primary morphological criteria and used in development of high-throughput assays. Our preliminary data suggests that the toxicity from NRTIs may be mediated through mitochondria. We observed mitochondrial energy failure associated with necrotic cell death. We will study the mechanism of this neurotoxicity by examining the roles of ATP generation, reactive oxygen species and mitochondria-specific heat-shock proteins. The ultimate goal of these studies will be to find compounds that may ameliorate the neurotoxicity of NRTIs. Nonimmunosuppressive neuroimmunophilin ligands will be used in this screening. Our screening program may yield clinically useful neuroprotective compounds that can be given in conjunction with the NRTIs as part of their HAART regimen. This is an ideal situation for a neuroprotective paradigm, because the neuroprotective drugs can be given in advance or in conjunction with the neurotoxic drugs. Furthermore, this assay system may be useful in development of NRTIs without the neurotoxic side effects.